1. Field of the Invention
The invention relates to one or more nucleic acids encoding a binding molecule specifically binding a human CD28 molecule, comprising                (a) a nucleic acid sequence encoding a VH region and a nucleic acid sequence encoding a VL region comprising CDRs in a human immunoglobulin framework, wherein                    (i) the CDRs of the VH region (CDR-H) comprise the amino acid sequences of SEQ ID NOS: 2 or 18 (CDR-H3), 4 or 20 (CDR-H2) and 6 or 22 (CDR-H1) or are encoded by the nucleic acid sequences of SEQ ID NOS: 1 or 17 (CDR-H3), 3 or 19 (CDR-H2) and 5 or 21 (CDR-H1); and            (ii) the CDRs of the VL region (CDR-L) comprise the amino acid sequences of SEQ ID NOS: 8 or 24 (CDR-L3), 10 or 26 (CDR-L2) and 12 or 28 (CDR-L1) or are encoded by the nucleic acid sequences of SEQ ID NOS: 7 or 23 (CDR-L3), 9 or 25 (CDR-L2) and 11 or 27 (CDR-L1); and                        (b) a nucleic acid sequence encoding a constant region of a human IgG1 or IgG4 antibody.        
2. Background Information
In the description a number of prior art documents including patent applications and manufacturer's instructions for use are mentioned. Whereas the disclosure content of these documents is not considered to be relevant for the patentability of the present invention, it is incorporated by reference into the present description.
The stimulation of resting T lymphocytes for activation, proliferation and functional differentiation requires the occupancy of two surface structures, so-called receptors: 1. of the antigen receptor having a different specificity from cell to cell and being necessary for the recognition of antigens, e.g. viral decomposition products; as well as 2. the CD28 molecule equally expressed on all resting T cells with the exception of a subgroup of the human CD8-T cells, the CD28 molecule naturally binding to ligands on the surface of other cells of the immune system. This is also called costimulation of the antigen-specific immune reaction by CD28. In cell culture, these processes can be simulated by occupancy of the antigen receptor as well as of the CD28 molecule with suitable monoclonal antibodies (mAb). In the classic system of costimulation neither the occupancy of the antigen receptor nor that of the CD28 molecule alone leads to T cell proliferation, however, the occupancy of both receptors is effective. This observation was made on T cells of the human, the mouse and the rat.
However, there are also known CD28-specific monoclonal antibodies (mAb) which may trigger T cell proliferation without costimulation. Such a superagonistic, i.e. the activation of resting T lymphocytes by CD28-specific mAb independent of the occupancy of the antigen receptor, is known for example from Tacke at al., Eur. J. Immunol. 1997, 27:239-247. This publication described two kinds of CD28 specific monoclonal antibodies having differing functional properties: costimulatory mAb which costimulate the activation of resting T cells only in case the antigen receptor is simultaneously occupied, and superagonistic mAb which can activate in vitro and in rats T lymphocytes of all classes to proliferate without occupying the antigen receptor.
Superagonistic monoclonal antibodies with specificity to the human CD28 molecule, which very efficiently activate and expand T cells in vitro without stimulation of the T cell antigen receptor (TCR), are further known from DE 101 60 516.1 as well as from Luhder et al., J. Exp. Med., 2003, 197: 955-966. However, in this work immobilzed antibodies were used, which, as shown further below, are not suitable for therapeutic use.
Furthermore, superagonistic anti-CD28 antibodies showed pronounced anti-inflammatory properties in animal models and in cell culture. Thus, for example, as documented by Schmidt et al., J. Neuroimmunol. 2003, 140: 143-152, the application of a superagonistic monoclonal antibody against the CD28 molecule of the rat may prevent the development of an inflammatory peripheral neuropathy, the Experimental Autoimmune Neuritis (EAN). From DE 102 12 108.7 as well as from Lin et al., Eur. J. Immunol., 2003, 33:626-638 it is known that superagonistic anti-CD28 antibodies may cause a superproportionally strong activation of regulatory T cells. The function of regulatory T cells is to control autoaggressive T cells and to make sure that generally no excessive inflammatory reaction develops (Schwartz, Nature Immunol., 2005, 6: 327-330). However, an intervention in CD28-mediated costimulation may shift the Th1/Th2 balance in favour of the pro-inflammatory Th1 phenotype and thus harbours the risk of aggravating autoimmune/inflammatory reactions (see Schmidt et al., supra).
For obvious reasons it is desirable that therapeutic antibody candidates prevent immunogenicity, i.e. triggering an immune response against the active substance, with the aim of fully exploiting the pharmacological activity in humans and simultaneously reducing undesirable side effects. In order to prevent the immunogenicity of not human-derived antibodies, for example, the “humanization” of antibodies by means of genetic engineering technology is state of the art. Here the antigen-binding site of an antibody originally not human-derived is conserved, while the rest of the antibody molecule, in particular the constant portions of the antibodies (constant domain or Fc fragment), is exchanged against a structurally related variant from the human genome (Hwang et al., Methods, 2005, 36:3-10).
As known from DE 102 30 223.5, the crosslinking of superagonistic anti-human CD28 antibodies enhances their ability to activate T lymphocytes in a cell suspension. For example, the proliferation of purified T lymphocytes is many times stronger when superagonistic antibodies are used in form of immobilized molecule complexes on paramagnetic beads instead of being present in soluble form in the T cell suspension. For galenical use in humans, the application of such complexed dosage forms of superagonistic anti-CD28 antibodies is, however, not possible for obvious reasons. Furthermore, in DE 102 30 223.5 anti-mouse-IgG antibodies immobilized on paramagnetic beads were used as crosslinking agent. In this respect, too, an analogous approach for a therapeutic application in humans is out of the question, since the use of anti-human-IgG antibodies has to be ruled out in view of the large number of cross-reactions to be expected. Last but not least, the approach described in DE 102 30 223.5 is limited to an in-vitro method wherein purified T cells are used. For the development of therapeutic superagonistic anti-CD28 antibodies it had therefore to be checked whether there exists a suitable antibody format which allows in vivo a sufficient crosslinking but does not lead to undesirable effects.
It is known that a natural recognition and crosslinking of Fc domains of antibodies in the human body are mediated by Fc receptors that are expressed on various cell types (Woof et al., Nature Reviews Immunol., 2004, 1-11). The aim of the Fc receptor-mediated antibody binding in a physiological context is the “removal” or destruction of antibody-occupied cells, since it must be assumed that antibodies are only formed against such cells that are derived from foreign tissue, that are bacterially or virally infected, are subject to stress or are malignantly degenerated. Important Fc receptor-mediated mechanisms for eliminating antibody-loaded cells are complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and opsonization, i.e. marking for phagocytosis by specialized phagocytes.
The Fc receptors responsible for the recognition of Fc domains of human antibodies of the IgG isotype may be classified into the groups CD64 (Fc gamma receptor I; high-affinity receptor); CD32 (Fc gamma receptor II; intermediate-affinity receptor) and CD16 (Fc gamma receptor III; low-affinity receptor). Their binding properties to antibodies of the IgG subgroups IgG1, IgG2, IgG3 and IgG4 as well as the effector functions triggered by the binding to these antibodies are known to a large extent (Woof et al., Nature Reviews Immunol., 2004, 1-11). Thus, the stimulation of Fc receptors binding to IgG1 or IgG4 (CD64 binds strongly to IgG1, moderately to IgG4; CD32 weakly to IgG1, not to IgG4; CD16b strongly to IgG1, very weakly or not at all to IgG4) generally also causes elimination of the target cells via ADCC or CDC.